Method of ultrasound imaging using microbubble-forming, solid X-ray contrast agents

ABSTRACT

Ultrasound contrast agents comprising a microbubble-generating suspension of a microparticulate X-ray contrast agent in a liquid carrier medium.

This application is a Division of application Ser. No. 08/170,197, filedFeb. 9, 1994, a 371 of PCT/EP92/01477, filed Jul. 3, 1992, now U.S. Pat.No. 5,607,661.

This invention relates to contrast agents of use in diagnosticultrasonic imaging.

It is well known that ultrasonic imaging comprises a potentiallyvaluable diagnostic tool, for example in studies of the vascular system,particularly in cardiography, and of tissue microvasculature. A varietyof contrast agents has been proposed to enhance the acoustic images soobtained, including suspensions of solid particles, emulsified liquiddroplets, gas bubbles and encapsulated gases or liquids. It is generallyaccepted that low density contrast agents which are easily compressibleare particularly efficient in terms of the acoustic backscatter theygenerate, and considerable interest has therefore been shown in thepreparation of gas-containing and gas-generating systems.

Initial studies involving free gas bubbles generated in vivo byintracardiac injection of physiologically acceptable substances havedemonstrated the potential efficiency of such bubbles as contrast agentsin echocardiography; such techniques are severely limited in practice,however, by the short lifetime of the free bubbles. Interest hasaccordingly been shown in methods of generating longer lived gasmicrobubble systems for use in echocardiography and other ultrasonicstudies.

One technique which has been proposed, for example in U.S. Pat. No.4,681,119, U.S. Pat. No. 4,442,843 and U.S. Pat. No. 1,657,756,comprises the injection of a suspension of a particulate solid(typically a saccharide such as Galactose) having a plurality ofgas-filled voids and preferably also a plurality of nuclei formicrobubble formation.

The present invention is based on our finding that microparticulateX-ray contrast agents may be administered in similar manner to providesubstantial enhancement of contrast in ultrasound studies such asechocardiography. While we do not wish to be bound by theoreticalconsiderations, it would appear that the particularly high density whichis characteristic of conventional X-ray contrast agents serves toenhance the density differential between the generated microbubbles andtheir surroundings, thereby improving the echogenicity of the system.

This use of X-ray contrast agents in suspension to generate microbubblesystems effective as ultrasound contrast agents may be contrasted withthe previously proposed use of sonicated solutions of X-ray contrastagents such as meglumine diatrizoate as preformed microbubble systemsand with previous proposals (e.g. as described in WO 90/07491) to usesimple suspensions of particles of insoluble X-ray contrast agents toenhance ultrasound images by virtue of reflection of ultrasound by theparticles themselves.

Thus according to one aspect of the present invention we provide X-raycontrast agents in microparticulate form adapted for administration as amicrobubble-generating suspension in an appropriate liquid carriermedium (e.g. sterile, pyrogen-free water for injection, orphysiologically saline), thereby acting as an ultrasound contrast agent.X-ray contrast agents may thus, for example, be presented in accordancewith the invention in the form of a pack comprising an and appropriateamount of microparticulate X-ray contrast agent and advantageously alsoa separate volume of liquid carrier, together with instructions forpreparing an intravenously administrable suspension of themicroparticulate X-ray contrast agent in the carrier liquid.

According to a further embodiment of the invention there is provided amethod of diagnosis in a human or animal subject by ultrasonic imagingwherein the contrast of the ultrasound image is enhanced by intravenousadministration of a microbubble-generating suspension of amicroparticulate X-ray contrast agent in an appropriate liquid carriermedium. A preferred method of diagnosis according to this embodiment ofthe invention is echocardiography.

The microparticulate X-ray contrast agents are advantageously presentedin the form of aggregates, for example having an aggregate size of20-125 micrometres, such as 30-50 micrometres, of particles having aparticle size of, for example, 1-50 micrometres, such as 5-10micrometres. Such aggregates, which may be prepared by, for example,conventional micronisation techniques such as grinding or milling, e.g.by ball-milling, will tend to contain a substantial volume of airadsorbed on their surfaces and entrained in voids such as interparticlecavities or at grain boundaries between the crystallites.

The particle size may, for example, be selected to be substantiallycommensurate with the desired microbubble size. In applications such asechocardiography this will typically be less than about 10 micrometres,preferably less than 7 micrometres, to permit passage through thepulmonary capillary bed and so allow enhanced ultrasound visualisationof the left side or the heart, preferably for more than one passage ofcirculation.

X-ray contrast agents which may be used in accordance with the inventioninclude the wide range of known X-ray contrast agents containingiodinated phenyl groups, for example the commercially availablecarboxylic acid and non-ionic amide X-ray contrast agents. Such agentstypically possess at least one 2,4,6-triiodophenyl group having at the3- and/or 5-positions groups selected from carboxyl, carbamoyl,N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino, N-alkylacylaminoand acylaminomethyl groupings. Alkyl groups present in such groupingsmay for example contain 1-6 carbon atoms; acyl groups present may forexample be alkanoyl groups containing up to 6 carbon atoms. Thusrepresentative acyl groups include acetyl, an example of anN-alkylacylamino group is N-methylacetamido, and representativeN-hydroxyalkylcarbamoyl groups include N-(1,3- and2,3-dihydroxypropyl)carbamoyl.

Examples of such X-ray contrast agents include carboxylic acids such asmetrizoic acid, diatrizoic acid, iothalamic acid or ioxaglic acid andsalts thereof. Non-ionic X-ray contrast agents include materials such asiohexol, iopentol, iopamidol, iodixanol, iopromide and metrizamide.Other agents include iodipamide, meglumine iodipamide, meglumineacetrizoate, meglumine diatrizoate, and acyloxyalkyl esters ofcarboxylic acids containing a triiodophenyl group, e.g. as described inGB-A-1363847, GB-A-2157283 and U.S. Pat. No. 4018783.

The use of water-soluble X-ray contrast agents comprises a preferredfeature of the present invention. It is thought that such agents producea longer-lasting ultrasound contrast effect since ongoing dissolution ofthe suspended water-soluble nicroparticulate material encouragescontinuing formation of microbubbles, which in turn may be stabilised bythe substantially saturated solution of X-ray contrast agent in theimmediate vicinity of the microbubbles tending to inhibit dissolution ofthe gas.

It may, however, be advantageous to modify the solubility properties ofsuch X-ray contrast agents by physically incorporating a lipid into oronto the microparticles, in order to provide products having propertiesparticularly suited to a specific application. There may also beadvantages in modifying insoluble X-ray contrast agents in similar waysand thus according to a further feature of the invention we providelipophile-carrying microparticulate X-ray contrast agents as a novelgeneral class of materials.

Lipids which may be admixed with microparticulate X-ray contrast agentsin accordance with the invention include fatty acids and monohydricalcohol esters thereof, fixed oils, fats, waxes, sterols, phospholipidsand glycolipids. The lipid may, for example, be a high molecular weight(e.g. C₁₀₋₅₀) straight chain saturated or unsaturated aliphatic acid,such as capric, palmitic, stearic, linolenic, behenic, docosanedioic ormelissic acid; an aralkanoic acid, e.g. a phenyl lower alkanoic acidsuch as 2-phenylbutyric acid; a triglyceride, for example a glycerylester of a high molecular weight (e.g. C₁₀₋₅₀) aliphatic acid, such asglyceryl trilaurate or glyceryl trimyristate; a cholanic acid such as5β-cholanic acid; a partially hydrogenated vegetable oil such ascottonseed oil or soyabean oil; a wax, for example beeswax or carnaubawax; a high molecular weight (e.g. C₁₀₋₅₀) straight chain aliphaticalcohol such as stearyl alcohol or cetyl alcohol; or a mixture thereof.Mixtures of high molecular weight fatty acids such as mixtures ofstearic and palmitic acids, mixtures of high molecular weight straightchain aliphatic alcohols, such as cetostearyl alcohol, mixtures ofpartially hydrogenated cottonseed and soyabean oils and mixtures of highmolecular weight aliphatic acids and glyceryl esters such as a mixtureof stearic acid and glyceryl trilaurate may, for example, be used.

Where it is desired to apply the lipid as a coating this may be effectedby, for example, slurrying the microparticulate X-ray contrast agent ina solution of the lipid in an organic solvent in which the X-raycontrast agent is substantially insoluble and thereafter removing thesolvent, e.g. by conventional means.

Alternatively the lipid ray be physically admixed with or within themicroparticulate X-ray contrast agent, using any convenient method.

In one preferred method according to the invention, which leads to thelipid being admixed within the microparticulate structure, the X-raycontrast agent and the lipid are each dissolved in appropriate mutuallymiscible solvents (e.g. water in the case of water-soluble X-raycontrast agents and a lower alkanol such as ethanol in the case oflipids such as fatty acids), the resulting solutions are mixed, thesolvents are removed (e.g. by evaporation under reduced pressure), and,if necessary, the resulting solid mixture is micronised (e.g. byconventional techniques such as grinding or milling, advantageously byball-milling, to yield the desired microparticles. It will beappreciated that all such operations should be effected under sterileconditions.

In general the lipid content of contrast agents according to theinvention man, for example, be in the range 0.01-5.0% w/w,advantageously 0.1-2.0% w/w, relative to the microparticulate X-raycontrast agent.

In addition to or alternatively to air, any other biocompatible gas maybe employed in the contrast agents of the invention, for examplenitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium,argon, sulphur hexafluoride and low molecular weight optionallyfluorinated hydrocarbons such as methane, acetylene or carbontetrafluoride. The term "gas" as used herein includes any substance inthe gaseous form at 37° C. The gas may be contained in the contrastagent in such a way that before use the product is non-contrast givingbut becomes effective on administration, e.g. as a result of the gasforming microbubbles as a soluble X-ray contrast agent dissolves. Therate of microbubble formation may thus be controlled by, for example,selection of an appropriate degree of lipid content. In general, any gasmay be introduced before, during or after any treatment such as lipidadmixture with the microparticulate X-ray contrast agent.

Gas precursors useful in contrast agents according to the inventioninclude carbonates and bicarbonates (e.g. sodium or ammoniumbicarbonate) and aminomalonate esters.

For applications in echocardiography, in order to permit free passagethrough the pulmonary system and to achieve resonance with the preferredimaging frequency of about 0.1-15 MHz, it may be convenient to employmicrobubbles and microparticles having an average size of 0.1-10 μm,e.g. 1-7 μm; the use of microparticles of average size 1-2 μm togenerate microbubbles with an average size of 4-7 μm is generallyadvantageous. Substantially larger bubbles and particles, e.g. withaverage sizes of us to 500 μm, may however be useful in otherapplications, for example gastrointestinal imaging.

The Invention is illustrated by the following Examples:

EXAMPLE 1 METRIZAMIDE

3.75 g of freeze-dried metrizamide containing 1.2 mg sodium calciumedetate (commercially available as Amipaque, NYCOMED AS, Norway) isfilled into a 20 ml vial. 3.75 g of the carrier liquid, consisting of 10ml sterile propylene glycol mixed with 90 ml of 5% sterile dextrosesolution, is then added and the resulting mixture is then shakenvigorously for 1-2 minutes. The mixture will now release gasmicrobubbles in the particle size range of 1-100 μm, which can beobserved by light microscopy.

The mixture is to be used within 5 minutes.

EXAMPLE 2 IOHEXOL

20.0 g of iohexol (NYCOMED AS, Oslo) was ball-milled in an aluminiumball-mill with 3×1.5 cm diameter aluminium balls for 45 minutes. Theresulting powder mixture consists of crystals and aggregates of iohexol.0.75 g of the powdered mixture is then filled into a 20 ml vial. 3.0 gof the carrier liquid, which consists of 10 ml sterile propylenglycolmixed with 90 ml sterile 5% dextrose solution, is added to the vial, andvigorously shaken for about 1-2 minutes. Gas microbubbles, as observedby light microscopy, are formed. The bubble size is within the range of1-100 μm.

The mixture is to be used within 5 minutes.

EXAMPLE 3 IODIXANOL

20.0 q of iodixanol (NYCOMED AS) was ball-milled as described in Example2. When mixing the resulting powder with the carrier liquid described inExample 2 followed by vigorously shaking for 1-2 minutes, gasmicrobubbles (1-100 μm) are formed. The bubbles can be observed by lightmicroscopy.

The mixture is to be used within 5 minutes.

EXAMPLE 4 METRIZAMIDE COATED WITH STEARIC ACID

6.75 g of metrizamide containing 2.4 mg sodium calcium edetate wasball-milled as described in Example 2. 10.6 mg stearic acid (Merck) isdissolved in 200 mg ethanol, and mixed with 5.3 g of the ball-milledpowder.

3.75 g of the resulting mixture is then mixed with 3.75 ml of thecarrier liquid described in Example 1 and shaken vigorously for 1-2minutes. The mixture will now release gas microbubbles in the particlesize range of 1-100 μm, which can be observed by light microscopy.

The mixture is to be used within 5 minutes.

EXAMPLE 5 METRIZAMIDE ADMIXED WITH PALMITIC ACID

10.0 g of freeze-dried metrizamide (NYCOMED AS, Oslo) were dissolved in14.2 g of distilled water, sterile filtered and then cooled on ice to atemperature of 4°-8° C. 0.2 g of palmitic acid was dissolved in 1.2 g of96% ethanol at 60° C. and sterile filtered. The fatty acid solution wasadded to the cold solution of the X-ray contrast agent under stirring,and the whole mixture was evaporated to dryness under vacuum (10 torr,40° C.). The product was dried in a desiccator overnight. The drysubstance was then ground using a stainless steel ball-mill (Retschcentrifugal ball-mill, S1) with a 50 ml grinding cup and 3×20 rim ballsfor 10 minutes under aseptic conditions. The-ground product was finallydried in a desiccator for 24 h before in vitro ultrasound measurementswere undertaken.

EXAMPLE 6 ECHOGENICITY IN VITRO

10 ml of propylene glycol mixed with 90 ml of 5% dextrose in water wasused as a carrier liquid for determining the echogenicity of products ofthe Examples. 1.0 g of each product to be tested was dispersed in 3.0 mlof the carrier liquid and shaken for 15 seconds. The resulting mixtureas added to 52 ml of 5% human serum albumin infusion solution in themeasurement cell and the acoustic effects of the products wereinvestigated by measuring the acoustic transmission through the samplesusing a 5 MHz broadband transducer in a pulse-reflection technique. Thetemperature in the measurement cell was stabilised to 37° C. andcirculation of the liquid was maintained by means of stirring at aconstant rate. Ultrasound transmission through the samples was measuredas a function of time over a duration of 390 seconds. Results werenormalized to measurements on a reference consisting of 55 ml of 5%human serum albumin infusion solution.

The products of the Examples generally showed higher echogenicity thanthe reference. The product of Example 5 was characterised by substantialduration of activity as evidenced by the half life of its attenuativeeffect.

I claim:
 1. A method of diagnosis in a human or animal subject byultrasonic imaging wherein contrast in the ultrasound image is enhancedby intravenous administration of an ultrasound contrast agentcomposition comprising aggregates of microparticles of a water-solublesolid X-ray contrast agent, said aggregates forming microbubbles ofbiocompatible gas when suspended in a liquid carrier medium.
 2. A methodof diagnosis as claimed in claim 1, wherein said ultrasound contrastagent composition comprises aggregates of microparticles of a lipid anda water-soluble X-ray contrast agent.
 3. A method as claimed in claim 2,wherein the aggregates have a size of 20-125 micrometers and themicroparticles have a size of 1-50 micrometers.
 4. A method as claimedin claim 2, wherein the aggregates have a size of 30-50 micrometers andthe microparticles have a size of 5-10 micrometers.
 5. A method asclaimed in claim 2, wherein the lipid content is 0.01-5.0% w/w relativeto the X-ray contrast agent.
 6. A method as claimed in claim 2, whereinthe lipid content is 0.01-2.0% w/w relative to the X-ray contrast agent.7. A method as claimed in claim 2, wherein the lipid is a C₁₀₋₅₀straight chain saturated or unsaturated aliphatic acid, a triglycerideof such an acid, or a phospholipid.
 8. A method as claimed in claim 1,wherein said biocompatible gas is selected from the group consisting ofair, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium,argon, sulphurhexafluoride, low molecular weight hydrocarbons,fluorinated low molecular weight hydrocarbons, and mixtures of air withnitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium,argon, sulphurhexafluoride, low molecular weight hydrocarbons, orfluorinated low molecular weight hydrocarbons.
 9. A method as claimed inclaim 2, wherein said biocompatible gas is selected from the groupconsisting of air, nitrogen, oxygen, hydrogen, nitrous oxide, carbondioxide, helium, argon, sulphurhexafluoride, low molecular weighthydrocarbons, fluorinated low molecular weight hydrocarbons and mixturesof air with nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide,helium, argon, sulphurhexafluoride, low molecular weight hydrocarbons,or fluorinated low molecular weight hydrocarbons.